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1. A method of detecting Salmonella in a sample, the method comprising
contacting a sample with forward and reverse primers that specifically
bind a Salmonella nucleic acid molecule in the presence of a nicking
enzyme, dNTPs, a detectable probe and a polymerase under conditions
permissive for the isothermal amplification of the nucleic acid molecule,
and detecting a Salmonella amplicon in the sample, wherein the method
detects the target nucleic acid sequence:
TABLE-US-00024
(SEQ ID NO: 1)
5'-CACCGAAATACCGCCAATAAAGTTCACAAAGATAATAATGATG
CCG-3',

wherein "m" indicates the position of a modification, and a probe
comprising the nucleic acid sequence:
TABLE-US-00039
(SEQ ID NO: 5)
5'-CGCCTGTGAACTTTATTGGCG-3'.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation under 35 U.S.C. .sctn.120 of PCT
international application Serial No. PCT/US2015/027036, filed Apr. 22,
2015, designating the United States and published in English, which
claims priority to and the benefit of U.S. Provisional Patent Application
Ser. No. 62/110,268, filed Jan. 30, 2015. The entire contents of each of
these applications is hereby incorporated by reference herein.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been
submitted electronically in ASCII format and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Jan. 15, 2016, is
named 049224.1050US1(00124)_SL.txt and is 8,913 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Salmonella is one of the most common pathogens of foodborne disease
worldwide. It is responsible for a large number of infections in both
humans and animals. In fact, Salmonella causes approximately 93.8 million
human infections and 155,000 deaths annually worldwide. Salmonella
infections have been associated with eating foods, such as meat, eggs and
fresh produce, contaminated with animal or human feces. The main causes
of Salmonella illness are poultry and eggs. Recognizing the importance of
preventing the spread of Salmonella, in 2014 the US FDA challenged US
scientists to develop improved methods for detecting Salmonella. Current
methods for detecting Salmonella in food products is difficult, expensive
and time-consuming. Rapid and accurate detection methods are urgently
required to prevent Salmonella contaminated food products from entering
the animal or human food chain.

SUMMARY OF THE INVENTION

[0004] As described below, the present invention features rapid and
accurate methods for detecting Salmonella (e.g., in a food product,
environmental sample, biological sample or other material).

[0005] In one aspect, the invention features a method of detecting
Salmonella in a sample, the method involving contacting a sample with
forward and reverse primers that specifically bind a Salmonella nucleic
acid molecule in the presence of a nicking enzyme, dNTPs, a detectable
probe and a polymerase under conditions permissive for the isothermal
amplification of the nucleic acid molecule, and detecting a Salmonella
amplicon in the sample, where the method detects one of the following
target sequences:

[0006] In another aspect, the invention features a method for detecting
Salmonella in a sample, the method involving contacting the sample with
forward and reverse primers having the following sequences, respectively:

[0013] In various embodiments of the above aspects or any other aspect of
the invention delineated herein, the sample contains a food product,
environmental sample, biological sample or other material. In particular
embodiments of the above aspects, the food product is intended for animal
or human consumption. In other embodiments of the above aspects, the food
product is pet food intended for consumption by a companion animal. In
still other embodiments of the above aspects, the food product is or is
derived from produce, poultry, fish, or beef. In still other embodiments
of the above aspects, the environmental sample is a water, soil or sewage
sample. In still other embodiments of the above aspects, the biological
sample is feces or a blood sample. In still other embodiments of the
above aspects, the sample is a culture medium. In still other embodiments
of the above aspects, the Salmonella is selected from the group
consisting of S typhi, S paratyphi-A, S schottmuelleri, S choleraesuis, S
typhimurium and S enteritidis. In still other embodiments of the above
aspects, the forward and reverse primers are selected from forward
primers:

In still other embodiments of the above aspects, the probe contains a
fluorescent moiety and a quencher. In still other embodiments of the
above aspects, the fluorescent moiety is CalRed.sub.610nm and the
quencher is Black Hole Quencher2 (BHQ2). In still other embodiments of
the above aspects, the forward and reverse primers comprise the following
sequences, respectively:

In still other embodiments of the above aspects, the nicking enzyme is
any one or more of N.Bst9I, N.BstSEI, Nb.BbvCI(NEB),
Nb.Bpu10I(Fermantas), Nb.BsmI(NEB), Nb.BsrDI(NEB), Nb.BtsI(NEB),
Nt.AlwI(NEB), Nt.BbvCI(NEB), Nt.Bpu10I(Fermentas), Nt.BsmAI, Nt.BspD6I,
Nt.BspQI(NEB), Nt.BstNBI(NEB), and Nt.CviPII(NEB). In still other
embodiments of the above aspects, the polymerase is Bst DNA polymerase I
or Gst DNA polymerase I. In still other embodiments of the above aspects,
the method is used periodically to monitor a site selected from the group
consisting of a field, crop, herd, food processing facility, and food
handling facility for the presence of Salmonella. In still other
embodiments of the above aspects, the monitoring is conducted about every
1, 3, 6, 9, or 12 months. In still other embodiments of the above
aspects, the modification is selected from the group consisting of
2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-alkyl, 2'-allyl,
2'-O-[2-(methylamino)-2-oxoethyl], 2'-hydroxyl (RNA), 4'-thio,
4'-CH.sub.2--O-2'-bridge, 4'-(CH.sub.2).sub.2--O-2'-bridge, and
2'-O--(N-methylcarbamate).

[0014] Other features and advantages of the invention will be apparent
from the detailed description, and from the claims.

DEFINITIONS

[0015] Unless defined otherwise, all technical and scientific terms used
herein have the meaning commonly understood by a person skilled in the
art to which this invention belongs. The following references provide one
of skill with a general definition of many of the terms used in this
invention: Singleton et al., Dictionary of Microbiology and Molecular
Biology (2nd ed. 1994); The Cambridge Dictionary of Science and
Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.
Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The
Harper Collins Dictionary of Biology (1991). As used herein, the
following terms have the meanings ascribed to them below, unless
specified otherwise.

[0016] By "amplicon" is meant a polynucleotide generated during the
amplification of a polynucleotide of interest. In one example, the
amplicon comprises at least a portion of a Salmonella invA
polynucleotide.

[0017] By "base substitution" is meant a substituent of a nucleobase
polymer that does not cause significant disruption of the hybridization
between complementary nucleotide strands.

[0018] In this disclosure, "comprises," "comprising," "containing" and
"having" and the like can have the meaning ascribed to them in U.S.
patent law and can mean "includes," "including," and the like;
"consisting essentially of" or "consists essentially" likewise has the
meaning ascribed in U.S. patent law and the term is open-ended, allowing
for the presence of more than that which is recited so long as basic or
novel characteristics of that which is recited is not changed by the
presence of more than that which is recited, but excludes prior art
embodiments.

[0019] By "complementary" or "complementarity" is meant that a nucleic
acid can form hydrogen bond(s) with another nucleic acid sequence by
either traditional Watson-Crick or Hoogsteen base pairing. Complementary
base pairing includes not only G-C and A-T base pairing, but also
includes base pairing involving universal bases, such as inosine. A
percent complementarity indicates the percentage of contiguous residues
in a nucleic acid molecule that can form hydrogen bonds (e.g.,
Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,
6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the
first oligonucleotide being based paired to a second nucleic acid
sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and
100% complementary respectively). To determine that a percent
complementarity is of at least a certain percentage, the percentage of
contiguous residues in a nucleic acid molecule that can form hydrogen
bonds (e.g., Watson-Crick base pairing) with a second nucleic acid
sequence is calculated and rounded to the nearest whole number (e.g., 12,
13, 14, 15, 16, or 17 nucleotides out of a total of 23 nucleotides in the
first oligonucleotide being based paired to a second nucleic acid
sequence having 23 nucleotides represents 52%, 57%, 61%, 65%, 70%, and
74%, respectively; and has at least 50%, 50%, 60%, 60%, 70%, and 70%
complementarity, respectively). As used herein, "substantially
complementary" refers to complementarity between the strands such that
they are capable of hybridizing under biological conditions.
Substantially complementary sequences have 60%, 70%, 80%, 90%, 95%, or
even 100% complementarity. Additionally, techniques to determine if two
strands are capable of hybridizing under biological conditions by
examining their nucleotide sequences are well known in the art.

[0020] "Detect" refers to identifying the presence, absence or amount of
the analyte to be detected. In one embodiment, the analyte is a
Salmonella polynucleotide. In a working example, an assay of the
invention detects the presence of Salmonella in a matrix of the
invention.

[0021] By "detectable probe" is meant a composition that when linked to a
moiety of interest renders the latter detectable, via spectroscopic,
photochemical, biochemical, immunochemical, or chemical means. For
example, useful detectable moieties include radioactive isotopes,
magnetic beads, metallic beads, colloidal particles, fluorescent dyes,
electron-dense reagents, enzymes (for example, as commonly used in an
ELISA), biotin, digoxigenin, or haptens. In one embodiment, a detectable
probe is a molecular beacon.

[0022] By "food product" is meant any material intended for animal or
human consumption.

[0023] By "hybridize" is meant to form a double-stranded molecule between
complementary polynucleotide sequences (e.g., a gene described herein),
or portions thereof, under various conditions of stringency. (See, e.g.,
Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.
R. (1987) Methods Enzymol. 152:507). Hybridization occurs by hydrogen
bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding, between complementary nucleobases. For example, adenine
and thymine are complementary nucleobases that pair through the formation
of hydrogen bonds.

[0024] By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA,
RNA) that is free of the genes which, in the naturally-occurring genome
of the organism from which the nucleic acid molecule of the invention is
derived, flank the gene. The term therefore includes, for example, a
recombinant DNA that is incorporated into a vector; into an autonomously
replicating plasmid or virus; or into the genomic DNA of a prokaryote or
eukaryote; or that exists as a separate molecule (for example, a cDNA or
a genomic or cDNA fragment produced by PCR or restriction endonuclease
digestion) independent of other sequences. In addition, the term includes
an RNA molecule that is transcribed from a DNA molecule, as well as a
recombinant DNA that is part of a hybrid gene encoding additional
polypeptide sequence.

[0025] The terms "isolated," "purified," or "biologically pure" refer to
material that is free to varying degrees from components which normally
accompany it as found in its native state. "Isolate" denotes a degree of
separation from original source or surroundings. "Purify" denotes a
degree of separation that is higher than isolation. A "purified" or
"biologically pure" protein is sufficiently free of other materials such
that any impurities do not materially affect the biological properties of
the protein or cause other adverse consequences. That is, a nucleic acid
or peptide of this invention is purified if it is substantially free of
cellular material, viral material, or culture medium when produced by
recombinant DNA techniques, or chemical precursors or other chemicals
when chemically synthesized. Purity and homogeneity are typically
determined using analytical chemistry techniques, for example,
polyacrylamide gel electrophoresis or high performance liquid
chromatography. The term "purified" can denote that a nucleic acid or
protein gives rise to essentially one band in an electrophoretic gel. For
a protein that can be subjected to modifications, for example,
phosphorylation or glycosylation, different modifications may give rise
to different isolated proteins, which can be separately purified.

[0026] By "nicking agent" is meant a chemical entity capable of
recognizing and binding to a specific structure in double stranded
nucleic acid molecules and breaking a phosphodiester bond between
adjoining nucleotides on a single strand upon binding to its recognized
specific structure, thereby creating a free 3'-hydroxyl group on the
terminal nucleotide preceding the nick site. In preferred embodiments,
the 3' end can be extended by an exonuclease deficient polymerase.
Exemplary nicking agents include nicking enzymes, RNAzymes, DNAzymes, and
transition metal chelators.

[0027] As used herein, the term "nucleic acid" refers to
deoxyribonucleotides, ribonucleotides, or modified nucleotides, and
polymers thereof in single- or double-stranded form. The term encompasses
nucleic acids containing known nucleotide analogs or modified backbone
residues or linkages, which are synthetic, naturally occurring, and
non-naturally occurring, which have similar binding properties as the
reference nucleic acid, and which are metabolized in a manner similar to
the reference nucleotides. Examples of such analogs include, without
limitation, 2' modified nucleotides (e.g., 2'-O-methyl, 2'-F
nucleotides).

[0028] By "periodic" is meant at regular intervals. Periodic monitoring
includes, for example, a schedule of tests that are administered daily,
bi-weekly, bi-monthly, monthly, bi-annually, or annually.

[0029] By "polymerase-arresting molecule" is meant a moiety associated
with a polynucleotide template/primer that prevents or significantly
reduces the progression of a polymerase on the polynucleotide template.
Preferably, the moiety is incorporated into the polynucleotide. In one
preferred embodiment, the moiety prevents the polymerase from progressing
on the template.

[0030] By "polymerase extension" is meant the forward progression of a
polymerase that matches incoming monomers to their binding partners on a
template polynucleotide.

[0031] By "reference" is meant a standard or control condition.

[0032] Sequence identity is typically measured using sequence analysis
software (for example, Sequence Analysis Software Package of the Genetics
Computer Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or
PILEUP/PRETTYBOX programs). Such software matches identical or similar
sequences by assigning degrees of homology to various substitutions,
deletions, and/or other modifications. Conservative substitutions
typically include substitutions within the following groups: glycine,
alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. In an exemplary approach to determining the
degree of identity, a BLAST program may be used, with a probability score
between e.sup.-3 and e.sup.-100 indicating a closely related sequence.

[0033] By "specific product" is meant a polynucleotide product resulting
from the hybridization of primer oligonucleotides to a complementary
target sequence and subsequent polymerase mediated extension of the
target sequence.

[0034] By "specifically binds" is meant an oligonucleotide probe of the
invention that binds a polynucleotide of the invention, but which does
not substantially recognize and bind other polynucleotides in a sample,
for example, a biological sample.

[0035] By "substantially isothermal condition" is meant at a single
temperature or within a narrow range of temperatures that does not vary
significantly.

[0036] By "target nucleic acid molecule" is meant a polynucleotide to be
analyzed. Such polynucleotide may be a sense or antisense strand of the
target sequence. The term "target nucleic acid molecule" also refers to
amplicons of the original target sequence.

[0038] Unless specifically stated or obvious from context, as used herein,
the term "about" is understood as within a range of normal tolerance in
the art, for example within 2 standard deviations of the mean. About can
be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,
0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from
context, all numerical values provided herein are modified by the term
about.

[0039] The recitation of a listing of chemical groups in any definition of
a variable herein includes definitions of that variable as any single
group or combination of listed groups. The recitation of an embodiment
for a variable or aspect herein includes that embodiment as any single
embodiment or in combination with any other embodiments or portions
thereof.

[0040] Any compositions or methods provided herein can be combined with
one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 provides the sequence of the Salmonella invA gene (SEQ ID
NO: 22). Highlighted in gray are the target sequence regions used for
design of isothermal detection assays. The target sequence region of a
commercially available Salmonella invA assay used herein as a reference
assay is shown in bold, underlined letters.

[0046] FIG. 6, comprising FIGS. 6A and 6B, provides a comparison of
isothermal amplification plots obtained using a DNAble assay of the
invention (shown in FIG. 6B) and a commercially available NEAR assay
targeting invA (shown in FIG. 6A).

DETAILED DESCRIPTION OF THE INVENTION

[0047] As described below, the present invention features rapid and
accurate methods for detecting Salmonella in a sample (e.g., in a food
product, environmental sample, biological sample or other material).

[0048] The invention is based, at least in part, on the discovery that
Salmonella can be detected by assaying food, environmental (e.g., water,
soil, sewage or other waste product), biological sample (e.g., feces) or
other samples using an isothermal nicking amplification reaction.

Salmonella

[0049] Salmonella species are Gram-negative, flagellated facultatively
anaerobic bacilli. There are over 1800 known serovars which current
classification considers to be separate species. The most common human
and animal pathogens include, but are not limited to, S typhi, S
paratyphi-A, S schottmuelleri, S choleraesuis, S typhimurium and S
enteritidis. The most common animal reservoirs are chickens, turkeys,
pigs, and cows; dozens of other domestic and wild animals also harbor
these organisms.

[0050] Salmonellosis ranges clinically from the common Salmonella
gastroenteritis (diarrhea, abdominal cramps, and fever) to enteric fevers
(including typhoid fever) which are life-threatening febrile systemic
illness requiring prompt antibiotic therapy. Focal infections and an
asymptomatic carrier state occur. The most common form of salmonellosis
is a self-limited, uncomplicated gastroenteritis.

[0051] Pathogenic salmonellae ingested in food survive passage through the
gastric acid barrier and invade the mucosa of the small and large
intestine and produce toxins. Invasion of epithelial cells stimulates the
release of proinflammatory cytokines which induce an inflammatory
reaction. The acute inflammatory response causes diarrhea and may lead to
ulceration and destruction of the mucosa. The bacteria can disseminate
from the intestines to cause systemic disease.

Nucleic Acid Amplification Methods

[0052] Nucleic acid amplification technologies have provided a means of
understanding complex biological processes, detection, identification,
and quantification of Salmonella. The present invention provides for the
detection of Salmonella in a sample by amplifying the DNA in an
isothermal nicking amplification reaction and is designed to detect all
serovars of Salmonella.

[0053] The polymerase chain reaction (PCR) is a common thermal cycling
dependent nucleic acid amplification technology used to amplify DNA
consisting of cycles of repeated heating and cooling of the reaction for
DNA melting and enzymatic replication of the DNA using a DNA polymerase.
Real-Time quantitative PCR (qPCR) is a technique used to quantify the
number of copies of a given nucleic acid sequence in a biological sample.
Currently, qPCR utilizes the detection of reaction products in real-time
throughout the reaction and compares the amplification profile to the
amplification of controls which contain a known quantity of nucleic acids
at the beginning of each reaction (or a known relative ratio of nucleic
acids to the unknown tested nucleic acid). The results of the controls
are used to construct standard curves, typically based on the logarithmic
portion of the standard reaction amplification curves. These values are
used to interpolate the quantity of the unknowns based on where their
amplification curves compared to the standard control quantities.

[0055] Isothermal nicking amplification reactions have similarities to PCR
thermocycling. Like PCR, nicking amplification reactions employ
oligonucleotide sequences which are complementary to a target sequences
referred to as primers. In addition, nicking amplification reactions of
target sequences results in a logarithmic increase in the target
sequence, just as it does in standard PCR. Unlike standard PCR, the
nicking amplification reactions progress isothermally. In standard PCR,
the temperature is increased to allow the two strands of DNA to separate.
In nicking amplification reactions, the target nucleic acid sequence is
nicked at specific nicking sites present in a test sample. The polymerase
infiltrates the nick site and begins complementary strand synthesis of
the nicked target nucleotide sequence (the added exogenous DNA) along
with displacement of the existing complimentary DNA strand. The strand
displacement replication process obviates the need for increased
temperature. At this point, primer molecules anneal to the displaced
complementary sequence from the added exogenous DNA. The polymerase now
extends from the 3' end of the template, creating a complementary strand
to the previously displaced strand. The second oligonucleotide primer
then anneals to the newly synthesized complementary strand and extends
making a duplex of DNA which includes the nicking enzyme recognition
sequence. This strand is then liable to be nicked with subsequent strand
displacement extension by the polymerase, which leads to the production
of a duplex of DNA which has nick sites on either side of the original
target DNA. Once this is synthesized, the molecule continues to be
amplified exponentially through replication of the displaced strands with
new template molecules. In addition, amplification also proceeds linearly
from each product molecule through the repeated action of the nick
translation synthesis at the template introduced nick sites. The result
is a very rapid increase in target signal amplification; much more rapid
than PCR thermocycling, with amplification results in less than ten
minutes.

Nicking Amplification Assays

[0056] The invention provides for the detection of Salmonella target
nucleic acid molecules amplified in an isothermal nicking amplification
assay. Such assays are known in the art and described herein. See, for
example, US Patent Application Publication 2009/0081670, PCT Application
2009/012246, and U.S. Pat. Nos. 7,112,423 and 7,282,328, each of which is
incorporated herein in its entirety. Polymerases useful in the methods
described herein are capable of catalyzing the incorporation of
nucleotides to extend a 3' hydroxyl terminus of an oligonucleotide (e.g.,
a primer) bound to a target nucleic acid molecule. Such polymerases
include those that are thermophilic and/or those capable of strand
displacement. In one embodiment, a polymerase lacks or has reduced 5'-3'
exonuclease activity and/or strand displacement activity. DNA polymerases
useful in methods involving primers having 2'-modified nucleotides at the
3' end include derivatives and variants of the DNA polymerase I isolated
from Bacillus stearothermophilus, also taxonomically re-classified as
Geobacillus stearothermophilus, and closely related thermophilic
bacteria, which lack a 5'-3' exonuclease activity and have
strand-displacement activity. Exemplary polymerases include, but are not
limited to the fragments of Bst DNA polymerase I and Gst DNA polymerase
I.

[0057] A nicking enzyme binds double-stranded DNA and cleaves one strand
of a double-stranded duplex. In the methods of the invention, the nicking
enzyme cleaves the top stand (the strand comprising the 5'-3' sequence of
the nicking agent recognition site). In a particular embodiment of the
invention disclosed herein, the nicking enzyme cleaves the top strand
only and 3' downstream of the recognition site. In exemplary embodiments,
the reaction comprises the use of a nicking enzyme that cleaves or nicks
downstream of the binding site such that the product sequence does not
contain the nicking site. Using an enzyme that cleaves downstream of the
binding site allows the polymerase to more easily extend without having
to displace the nicking enzyme. Ideally, the nicking enzyme is functional
under the same reaction conditions as the polymerase. Exemplary nicking
enzymes include, but are not limited to, N.Bst9I, N.BstSEI,
Nb.BbvCI(NEB), Nb.Bpu10I(Fermantas), Nb.BsmI(NEB), Nb.BsrDI(NEB),
Nb.BtsI(NEB), Nt.AlwI(NEB), Nt.BbvCI(NEB), Nt.Bpu10I(Fermentas),
Nt.BsmAI, Nt.BspD6I, Nt.BspQI(NEB), Nt.BstNBI(NEB) and Nt.CviPII(NEB).
Sequences of nicking enzyme recognition sites are provided at Table 1.

Nicking enzymes also include engineered nicking enzymes created by
modifying the cleavage activity of restriction endonucleases (NEB
expressions July 2006, vol 1.2). When restriction endonucleases bind to
their recognition sequences in DNA, two catalytic sites within each
enzyme for hydrolyzing each strand drive two independent hydrolytic
reactions which proceed in parallel. Altered restriction enzymes can be
engineered that hydrolyze only one strand of the duplex, to produce DNA
molecules that are "nicked" (3'-hydroxyl, 5'-phosphate), rather than
cleaved. Nicking enzymes may also include modified CRISPR/Cas proteins,
Transcription activator-like effector nucleases (TALENs), and Zinc-finger
nucleases having nickase activity.

[0058] A nicking amplification reaction typically comprises nucleotides,
such as, for example, dideoxyribonucleoside triphosphates (dNTPs). The
reaction may also be carried out in the presence of dNTPs that comprise a
detectable moiety including but not limited to a radiolabel (e.g.,
.sup.32P, .sup.33P, .sup.125I, .sup.35S) an enzyme (e.g., alkaline
phosphatase), a fluorescent label (e.g., fluorescein isothiocyanate
(FITC)), biotin, avidin, digoxigenin, antigens, haptens, or
fluorochromes. The reaction further comprises certain salts and buffers
that provide for the activity of the nicking enzyme and polymerase.

[0059] This invention provides methods of monitoring a nicking
amplification reaction in real time, utilizing the amplification strategy
as described above. In one embodiment, quantitative nucleic acid
amplification utilizes target nucleic acids amplification alongside a
control amplification of known quantity. The amount of target nucleic
acid can be calculated as an absolute quantification or a relative
quantification (semi-quantitative) based on the source of the control
(exogenous or endogenous control).

[0060] Quantification of the unknown nucleotide sequence can be achieved
either through comparison of logarithmic threshold amplification of the
unknown to a series of known target sequences in either a separate set of
reactions or in the same reaction; or as an internal endogenous or
exogenous co-amplification product which produces a threshold value,
indicative of either a positive result (if the unknown exceeds the
threshold) or negative result (if the unknown does not exceed the
threshold).

3' Recognition Region

[0061] The invention provides a primer having a 3' recognition sequence
whose primer-target formation is stable and has the potential to enhance
Salmonella nucleic acid amplification reaction performance. The 3'
recognition region specifically binds to the Salmonella nucleic acid
molecule, for example a complementary sequence of the Salmonella nucleic
acid molecule.

[0062] In particular, a primer of the invention having a 3' recognition
sequence is useful in nicking amplification assays. Additionally, the
Salmonella target specific 3' recognition region comprises one or more 2'
modified nucleotides (e.g., 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro,
2'-alkyl, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 2'-hydroxyl (RNA),
4'-thio, 4'-CH.sub.2--O-2'-bridge, 4'-(CH.sub.2).sub.2--O-2'-bridge, and
2'-O--(N-methylcarbamate)). Without being bound to theory, it is
hypothesized that incorporating one or more 2' modified nucleotides in
the recognition regions reduces or eliminates intermolecular and/or
intramolecular interactions of primers/templates (e.g., primer-dimer
formation), and, thereby, reduces or eliminates the background signal in
isothermal amplification. The 2' modified nucleotide preferably has a
base that base pairs with the target sequence. In particular embodiments,
two or more 2' modified nucleotides (e.g., 2, 3, 4, 5 or more 2' modified
nucleotides) in the Salmonella target specific recognition region are
contiguous (e.g., a block of modified nucleotides). In some embodiments,
the block of 2' modified nucleotides is positioned at the 3' end of the
target specific recognition region. In other embodiments, the block of 2'
modified nucleotides is positioned at the 5' end of the Salmonella target
specific recognition region. When the block of 2' modified nucleotides is
positioned at the 5' end of the target specific recognition region, the
2' modified nucleotides may be separated from the nick site by one or
more non-modified nucleotides (e.g., 2, 3, 4, 5 or more 2' unmodified
nucleotides). Applicants have found that positioning of one or more 2'
modified nucleotides or of a block of 2' modified nucleotides alters the
kinetics of amplification. When the one or more 2' modified nucleotides
or block of 2' modified nucleotides are positioned at or near the 5' end
of the recognition region or proximal to the nick site, real-time
amplification reactions showed decreased time to detection. Additionally,
the signal curve is contracted and the slope of the curve shifted.

[0063] In a related embodiment, ratios of a primer having one or more 2'
modified nucleotides can be used to alter the time-to-detection and/or
the efficiency of the reaction for the `tuning` of reactions, resulting
in a predictable control over reaction kinetics. Increasing the ratio of
primer having one or more 2' modified nucleotides at the 3' end of the
recognition sequence to primer having one or more 2' modified nucleotides
at the 5' end of the recognition sequence contracted the signal curve and
shifted the slope of the curve. It is advantageous to be able to "tune" a
reaction providing a means to manipulate both the time-to-detection as
well as the efficiency of the reaction. Relative quantification using an
internal control requires that two important conditions be met. First, it
is beneficial to be able to modify a reaction's time-to-detection
creating a non-competitive reaction condition. Thus, by affecting the
control reaction to be detectable at a later time-point (relative to the
target of interest) the control reaction does not out-compete the
specific target of interest even when the target of interest is in low
initial abundance. Second, to ensure a true relative abundance
calculation, it is required that the control and specific target
reactions have matched efficiencies. By controlling the efficiency of
each reaction using a "tuning" condition enables reactions to be matched
allowing for satisfactory relative quantification calculations. Tuning
the reactions can be used to match efficiencies of target nucleic acid
amplification and reference nucleic amplification (e.g., internal
standard) in quantitative PCR (qPCR). Additionally, amplification curves
of the target nucleic acid and the internal standard may be altered so
time of detection of their amplification products are separated, while
providing the same efficiency for target nucleic acid amplification and
internal standard amplification. Through the use of specific combinations
and ratios of oligonucleotide structures within a reaction it is possible
to create conditions which enable tuned reaction performance.

Target Nucleic Acid Molecules

[0064] Methods and compositions of the invention are useful for the
amplification and/or identification of a Salmonella nucleic acid molecule
in a test sample. The target sequences is amplified from virtually any
sample that comprises a Salmonella nucleic acid molecule.

[0065] Exemplary test samples include environmental samples, agricultural
products or other foodstuffs, and their extracts, body fluids (e.g.
blood, serum, plasma, feces, or gastric fluid), tissue extracts, and
culture media (e.g., a liquid in which a cell, such as a pathogen cell,
has been grown). If desired, the sample is purified prior to inclusion in
a nicking amplification reaction using any standard method typically used
for isolating a nucleic acid molecule from a biological sample.

[0066] In one embodiment, primers amplify a target nucleic acid of a
pathogen to detect the presence of Salmonella in a sample. Methods of the
invention provide for the detection of 50 copies per reaction are
detected Salmonella in a sample.

Applications

[0067] Target nucleic acid amplification using primers of the invention
have characteristics useful for rapid detection of Salmonella nucleic
acid molecules. Compositions and methods of the invention are
particularly useful for the detection of contaminated food products,
where a rapid answer is desired (e.g., detectable amplification in under
15, 10, 9, 8, 7, 6, 5 minutes or less).

[0068] In particular embodiments, the invention provides for the use of a
Salmonella nicking amplification reaction assay in the field, in
containers for transport, in warehouses, grain elevators, food processing
facilities, grocery stores, restaurants, kitchens, or any other venue
where food is handled, stored, or prepared for human or animal
consumption. In other embodiments, the sample is an environmental sample,
including but not limited to, water, soil, waste product (e.g., feces),
boot swabs, or sewage. In particular embodiments, the invention is useful
for assaying a poultry and birds (e.g., chicken, turkey, geese, ducks,
wild flocks), for facilities where poultry is processed (e.g., slaughter
house, coops) and from poultry derived food stuffs, including eggs and
egg products (e.g., egg whites). In other embodiments, the invention
provides for the use of nicking amplification reaction assays in field
work, where access to thermocycling equipment is unavailable or would be
prohibitively expensive. In still other embodiments, the invention
provides for the use of nicking amplification reaction assays in a
setting where rapid quantitative answers are desired.

Detectable Oligonucleotide Probes

[0069] The present invention provides for the quantitative detection of
target nucleic acid molecules or amplicons thereof in a nicking
amplification reaction using non-amplifiable detectable polynucleotide
probes comprising at least one polymerase-arresting molecule (e.g.,
nucleotide modification or other moiety (e.g., quencher, fluorescent
moiety) that renders the oligonucleotide capable of binding a target
nucleic acid molecule, but incapable of supporting template extension
utilizing the detectable oligonucleotide probe as a target). Without
wishing to be bound by theory, the presence of one or more moieties which
does not allow polymerase progression likely causes polymerase arrest in
non-nucleic acid backbone additions to the oligonucleotide or through
stalling of a replicative polymerase (i.e. C3-spacer, damaged DNA bases,
other spacer moiety, O-2-Me bases). These constructs thus prevent or
reduce illegitimate amplification of the probe during the course of a
nicking amplification reaction. This distinguishes them from conventional
detection probes, which must be added at the end of the nicking
amplification reaction to prevent their amplification.

[0070] Conventional detection probes have proven impractical for
quantitating a nicking amplification reaction in real time. If
conventional detection probes are incorporated into the nicking
amplification reaction, these conventional detection probes are amplified
concurrently with the target. The amplification of these detection
molecules masks the detection of legitimate target amplicons due to the
number of starting molecules of the detection probe at the start of the
reaction.

[0071] The invention provides non-amplifiable detectable polynucleotide
probe that comprise at least one polymerase-arresting molecule. A
polymerase-arresting molecule of the invention includes, but is not
limited to, a nucleotide modification or other moiety that blocks
template extension by replicative DNA polymerases, thereby preventing the
amplification of detection molecules; but can allow proper hybridization
or nucleotide spacing to the target molecule or amplified copies of the
target molecule. In one embodiment, a detectable oligonucleotide probe of
the invention comprises a 3 carbon spacer (C3-spacer) that prevents or
reduces the illegitimate amplification of a detection molecule.

[0072] In one embodiment, a detectable oligonucleotide probe comprises one
or more modified nucleotide bases having enhanced binding affinity to a
complementary nucleotide. Examples of modified bases include, but are not
limited to 2' Fluoro amidites, and 2'OMe RNA amidites (also functioning
as a polymerase arresting molecule). Detectable oligonucleotide probes of
the invention can be synthesized with different colored fluorophores and
may be designed to hybridize with virtually any target sequence. In view
of their remarkable specificity, a non-amplifiable detectable
polynucleotide probe of the invention is used to detect a single target
nucleic acid molecule in a sample, or is used in combination with
detectable oligonucleotide probes each of which binds a different target
nucleic acid molecule. Accordingly, the non-amplifiable detectable
polynucleotide probes of the invention may be used to detect one or more
target nucleic acid molecules in the same reaction, allowing these
targets to be quantitated simultaneously. The present invention
encompasses the use of such fluorophores in conjunction with the
detectable oligonucleotide probes described herein.

Kits

[0073] The invention also provides kits for the detection of a target
Salmonella nucleic acid molecule. Such kits are useful for the detection
or quantitation of a target Salmonella nucleic acid in a sample (e.g.,
food product, environmental sample, biological sample or other material).
Kits of the present invention may comprise, for example, one or more
polymerases, forward and reverse primers, and one or more nicking
enzymes, and a detectable probe as described herein. Where one target is
to be amplified, one or two nicking enzymes may be included in the kit.

[0074] The kits of the present invention may also comprise one or more of
the components in any number of separate containers, packets, tubes
(e.g., <0.2 ml, 0.2 ml, 0.6 ml, 1.5 ml, 5.0 ml, >5.0 ml), vials,
microtiter plates (e.g., <96-well, 96-well, 384-well, 1536-well,
>1536-well), ArrayTape, and the like, or the components may be
combined in various combinations in such containers. In various
embodiments, the kit further comprises a pair of primers capable of
binding to and amplifying a reference sequence that can be used as a
positive control. In yet other embodiments, the kit comprises a sterile
container which contains the primers; such containers can be boxes,
ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other
suitable container form known in the art. Such containers can be made of
plastic, glass, laminated paper, metal foil, or other materials suitable
for holding nucleic acids.

[0075] The components of the kit may, for example, be present in one or
more containers, for example, all of the components may be in one
container, or, for example, the enzymes may be in a separate container
from the primers. The components may, for example, be dried (e.g.,
powder) or in a stable buffer (e.g., chemically stabilized, thermally
stabilized). Dry components may, for example, be prepared by
lyophilization, vacuum and centrifugal assisted drying and/or ambient
drying. In various embodiments, the polymerase and nicking enzymes are in
lyophilized form in a single container, and the primers are either
lyophilized, freeze dried, or in buffer, in a different container. In
some embodiments, the polymerase, nicking enzymes, and the primers are,
in lyophilized form, in a single container. In other embodiments, the
polymerase and the nicking enzyme may be separated into different
containers.

[0076] Kits may further comprise, for example, dNTPs used in the reaction,
or modified nucleotides, cuvettes or other containers used for the
reaction, or a vial of water or buffer for re-hydrating lyophilized
components. The buffer used may, for example, be appropriate for both
polymerase and nicking enzyme activity.

[0077] The kits of the present invention may also comprise instructions
for performing one or more methods described herein and/or a description
of one or more compositions or reagents described herein. Instructions
and/or descriptions may be in printed form and may be included in a kit
insert. A kit also may include a written description of an Internet
location that provides such instructions or descriptions.

[0078] The practice of the present invention employs, unless otherwise
indicated, conventional techniques of molecular biology (including
recombinant techniques), microbiology, cell biology, biochemistry and
immunology, which are well within the purview of the skilled artisan.
Such techniques are explained fully in the literature, such as,
"Molecular Cloning: A Laboratory Manual", second edition (Sambrook,
1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture"
(Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental
Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells"
(Miller and Calos, 1987); "Current Protocols in Molecular Biology"
(Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994);
"Current Protocols in Immunology" (Coligan, 1991). These techniques are
applicable to the production of the polynucleotides and polypeptides of
the invention, and, as such, may be considered in making and practicing
the invention. Particularly useful techniques for particular embodiments
will be discussed in the sections that follow.

[0079] The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and description of
how to make and use the assay, screening, and therapeutic methods of the
invention, and are not intended to limit the scope of what the inventors
regard as their invention.

EXAMPLES

Example 1

Test Kit for Qualitative Detection of DNA from Salmonella

[0080] Rapid, point of need detection of Salmonella is required to effect
interventions to prevent its spread. A test kit was generated for
qualitative detection of DNA from Salmonella. The sequence of the invA
target gene is provided in FIG. 1. The detection assay is based on an
isothermal nucleic acid amplification method. Test samples were prepared
from simulated pet food or enriched culture. 1 mL of enriched sample was
added to a microcentrifuge tube and centrifuged at 10,000.times.G for 5
minutes. The supernatant was discarded and the pellet was suspended in
100 .mu.L of a buffer comprising Tris-Hcl, Magnesium Sulfate, Sodium
Sulfate, Ammonium Sulfate, and Triton X 100. The sample and buffer are
heated to 95.degree. C. for 5 minutes then centrifuged to pellet the
debris. The supernatant was diluted 1:10 in the buffer, and 5 .mu.l of
crude prep was diluted in 50 .mu.l buffer. The amplification reaction was
run at 56.degree. C. The amplification reaction contains excess of
forward primer (e.g., 600, 800 nM), 100-200 nM reverse primer, 300-400 nM
probe, 250-300 nM dNTPs, 10-20 Units a Bst DNA polymerase I, and 7-8
units of nicking enzyme Nt.BstNBI(NEB), dNTPs. The sequences of primers
and probes is provided at FIGS. 2 and 3.

[0081] The amplification and detection reactions displayed a high signal
to noise ratio, early onset of exponential amplification, steep
amplification slope, rapid time to detection, and low signal variance
among replicated assay reactions. All target control samples showed
robust signal. The assay was further tested and detected a list of over
100 Salmonella serotypes. These results indicate that the provides
compositions and methods for the rapid and sensitive detection of
Salmonella.

Example 2

Analytical Limit of Detection (ALOD) of Salmonella

[0082] FIG. 4 shows isothermal amplification plots of a target genomic DNA
dilution series of DNAble assay reactions targeting region 1 of the
"invA" gene carried out on a LC480 thermocycler from Roche Diagnostics
Inc. The target-specific probe signal was detected in the 533-610 nm
fluorescence channel. All reactions were set-up in 50 .mu.l volume using
800 nmol of the forward primer, 200 nmol of the reverse primer and 400
nmol of the molecular beacon probe under reaction conditions described
herein above. In sets of three technical replicates for each copy number,
various amounts of purified Salmonella enterica genomic DNA ranging from
0 copies per reaction (no target control reactions, blue amplification
plots), 50 copies per reaction (yellow amplification plots), 500 copies
per reaction (black amplification plots), 5000 copies per reaction
(purple amplification plots), 50,000 copies per reaction (yellow
amplification plots) and 500,000 copies per reaction (red amplification
plots) were added to the reaction. Reliable detection of the region I
"invA" target is demonstrated down to 50 copies per 50 .mu.l reaction.

Example 3

Biological Limit of Detection (BLOD) of Salmonella

[0083] FIG. 5 shows isothermal amplification plots of DNAble assay
reactions targeting region I of the "invA" gene carried out on a LC480
thermocycler from Roche Diagnostics Inc. A target-specific probe signal
was detected in the 533-610 nm fluorescence channel. All reactions were
set-up in 50 .mu.l volume using 800 nmol of the forward primer with the
optimal 5'-tail combination, 200 nmol of the reverse primer with optimal
5'-tail combination and 400 nmol of the molecular beacon probe under
standard reaction conditions described herein above. In sets of three
technical replicates, crude samples containing Salmonella enterica
genomic DNA extracted from bacterial cultures inoculated with counts of
live Salmonella cells equivalent to colony forming units (CFU) ranging
from 0 CFU per reaction (NTC, i.e. no target control reactions, orange
amplification plots), 10.sup.4 CFU per reaction (black amplification
plots), 10.sup.5 CFU per reaction (turquoise amplification plots),
10.sup.6 CFU (brown amplification plots) and 10.sup.7 CFU per reaction
(yellow amplification plots) were added to the reaction. Reliable
detection of the region I "invA" target is demonstrated down to 10.sup.4
CFU per 50 .mu.l reaction.

Example 4

Salmonella Assay Comparison

[0084] FIG. 6 shows amplification plots of a commercially available
isothermal amplification assay based on the NEAR technology (FIG. 6A) and
amplification plots carried out using primers and probes (FIG. 2)
targeting region 1 of the "invA" gene (FIG. 6B). All reactions were
carried out on a LC480 thermocycler from Roche Diagnostics Inc. The
target-specific probe signal was detected in the 533-610 nm fluorescence
channel. Reactions of both assays were set-up in 50 .mu.l volume as
described elsewhere. In sets of two (FIG. 6A) and three (FIG. 6B)
technical replicates for each gDNA copy number, various amounts of
purified Salmonella enterica genomic DNA ranging from 0 copies per
reaction (NTC, i.e. no target control reactions, blue amplification
plots), 50 copies per reaction (yellow amplification plots), 500 copies
per reaction (black amplification plots), 5000 copies per reaction
(purple amplification plots), 50,000 copies per reaction (yellow
amplification n plots) and 500,000 copies per reaction (red amplification
plots) were added to the reaction. The commercial NEAR-based assay failed
to detect the "invA" target sequence at gDNA copy numbers
<5000/reaction.

Example 5

Specificity of the Salmonella Assay

[0085] Using the reaction conditions described herein above, the
Salmonella strains delineated below were tested with the DNAble 3.0
Salmonella assay. The assay successfully detected all of the Salmonella
strains identified in the "Inclusivity list." The assay did not
cross-react with the bacteria listed on the "Exclusivity list."

[0086] From the foregoing description, it will be apparent that variations
and modifications may be made to the invention described herein to adopt
it to various usages and conditions. Such embodiments are also within the
scope of the following claims.

[0087] The recitation of a listing of elements in any definition of a
variable herein includes definitions of that variable as any single
element or combination (or subcombination) of listed elements. The
recitation of an embodiment herein includes that embodiment as any single
embodiment or in combination with any other embodiments or portions
thereof.

[0088] All patents and publications mentioned in this specification are
herein incorporated by reference to the same extent as if each
independent patent and publication was specifically and individually
indicated to be incorporated by reference.